PILOT PROJECT 8: HUMORAL IMMUNITY TO A NOVEL PNEUMOCYSTIS CARINII ANTIGEN

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 1)重组蛋白表达载体的构建及表达。为了比较我们新的P55蛋白(P55-V3)和先前报道的P55蛋白(P55-V0)的抗原性，我们从辛辛那提大学的斯穆利安博士那里获得了P55-V0质粒构建。出乎意料的是，我们发现该构建体不表达重组蛋白。多个克隆的DNA序列分析显示，编码区中部的单个核苷酸A缺失，导致阅读框架发生变化，并在下游引入多个终止密码子。因此，我们自制了新的构建物，其中一个构建了全长P55-V0序列，另外三个重叠片段构建了对应于我们之前表达的P55-v3片段的构建体。 2)多肽的合成及抗血清的制备：我们从Sigma获得了1个针对P55-V0的合成肽和两个针对P55-V3的抗血清。该抗血清将用于研究p55-v3和p55-v0的表位定位和差异表达模式。 3)动物模型：选用40只大鼠，随机分为3组。A组(10只)为对照组，饲养在SPF环境中，不接受任何药物治疗。B组(20只大鼠)和C组(10只大鼠)共住在一个常规房间(无滤器盖的开放式笼子)。B组采用免疫抑制法诱发自发性卡氏肺炎性肺炎(PCP)。Depo-Medrol剂量为2 mg/100g体重，前4周皮下注射2次，后6周每周皮下注射1次。C组不接受免疫抑制，但B组自然暴露于卡氏肺孢子虫，免疫抑制3-4周后，B组大鼠全部发病，体重明显减轻。免疫抑制后8-10周有5只大鼠死亡。对这5只大鼠的肺部进行染色和聚合酶链式反应检测，均未发现卡氏肺孢子虫阳性。此外，我们还用整个卡氏肺孢子虫细胞裂解物进行了蛋白质印迹，并分析了免疫抑制后8-10周大鼠的血清和对照组大鼠的血清。没有大鼠表现出阳性抗体反应。这些老鼠没有被感染的原因尚不清楚。最近，我们从其他研究人员那里获得了卡氏肺孢子虫活体，并通过气管接种了5只大鼠。目前还不能确定他们中是否有人被感染。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 1) Plasmid construction and expression of recombinant proteins. To compare the antigenicity of our new p55 protein (p55-v3) with previously described p55 protein (p55-v0), we obtained the p55-v0 plasmid construct from Dr. Smulian, University of Cincinnati. Unexpectedly, we found that this construct did not express recombinant protein. DNA sequence analysis of multiple clones revealed a deletion of a single nucleotide A in the middle of the coding region, resulting in reading frame changes and introduction of multiple stop codons downstream. Therefore, we made new constructs by ourselves, with one construct for the full-length p55-v0 sequence and additional constructs for three overlapping fragments, which are corresponding to the fragments of p55-v3 we had expressed before. 2) Synthesis of peptides and production of antisera: We have obtained synthetic peptides and antisera, one specific for p55-v0 and two specific for p55-v3, from Sigma. The antisera will be used to study the epitope mapping and differential expression patterns of p55-v3 and p55-v0 as proposed in Specific Aim 1. 3) Animal models: We used 40 rats divided into 3 groups. Group A (10 rats) was a control group and housed in SPF conditions and did not receive any medication. Groups B (20 rats) and C (10 rats) were cohoused together in a conventional room (open cages without filter tops). Group B received immunosuppression with Depo-medrol to provoke spontaneous P. carinii pneumoniae (PCP). The dose of Depo-Medrol used was 2 mg/100g body weight subcutaneously twice a week for the first 4 weeks, and once a week for another 6 weeks. Group C didnt receive immunosuppression but were expected to have natural exposure to P. carinii from group B. After 3-4 weeks of immunosuppression all rats in Group B became sick and had apparent weight loss. Five rats died 8-10 weeks after immunosuppression. Examination of the lungs of these 5 rats by staining and PCR found that none of them were positive for P. carinii. In addition we did western blot with whole P. carinii cell lysates and analyzed sera from rats obtained at 8-10 weeks after immunosuppression and sera from control rats. No rats showed positive antibody responses. The reasons why these rats were not infected are unknown. Very recently we obtained live P. carinii organisms from other investigators and did transtracheal inoculation with 5 rats. It has not been determined if any of them are infected.